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Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves
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Original Article
Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves Q5
Narasimharaju Kalidindi a,*, Nagarasanakote Venkataswamy Thimmaiah b, Nagepally Venkataramareddy Jagadeesh c, Ramaiah Nandeep a, Sathyanarayana Swetha a, Bharat Kalidindi d a
Department of Pharmacology, Visveswarapura Institute of Pharmaceutical Sciences, Bangalore, Karnataka, India Department of Pharmacology, KLE College of Pharmacy, Belgaum, Karnataka, India c Department of Pharmacology, SET's College of Pharmacy, Dharwad, Karnataka, India d SDM College of Ayurveda Kuthpady, Udupi, Karnataka, India b
article info
abstract
Article history:
An increasing demand for natural additives has shifted the attention from synthetic to
Received 1 November 2014
natural antioxidants and antifungal agents. This study was carried out to evaluate the
Received in revised form
antifungal and antioxidant activities of methanol, chloroform, and aqueous extracts of
15 April 2015
Annona squamosa Linn. leaves. The antifungal activities of all extracts of A. squamosa leaves
Accepted 26 April 2015
against five different strains of fungi (Alternaria alternata, Candida albicans, Fusarium solani,
Available online xxx
Microsporum canis, and Aspergillus niger) were evaluated by the agar well diffusion method and the minimum inhibitory concentration of each extract was assessed by antifungal suscep-
Q1
Keywords:
tibility using the broth microdilution method. The antioxidant potential of each extract was
acetogenins
determined by free radicals (1,1-diphenyl-2-picrylhydrazyl, nitric oxide, and hydrogen
Annona
peroxide) scavenging activity and reducing power property of A. squamosa leaves. Both
antifungal
organic and aqueous extracts were found to express dose-dependent inhibition against all
antioxidant
tested fungi strains in both agar well diffusion and broth dilution methods. The free radical
extract
scavenging activity and reducing power property of all extracts were found to be concentration dependent, with the methanol extract exhibiting higher antioxidant activity than the chloroform extract, which was more effective than the aqueous extract of A. squamosa leaves. Results of phytochemical analysis of extracts showed the presence of glycosides, saponins, tannins, flavonoids, phenols, etc. The results obtained from in vitro studies of antifungal and antioxidant activities clearly suggest that the methanol, chloroform, and aqueous extracts of A. squamosa leaves possess antifungal and antioxidant activity. Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.
* Corresponding author. Department of Pharmacology, Visveswarapura Institute of Pharmaceutical Sciences, 22nd Main, 24th Cross, Banashankari II Stage, Bangalore 560 070, Karnataka, India. E-mail address: [email protected] (N. Kalidindi). http://dx.doi.org/10.1016/j.jfda.2015.04.012 1021-9498/Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Introduction
In modern times, the use of herbal products has significantly increased in the developed countries as well as in several other countries. According to a World Health Organization estimate, 80% of the world's population presently uses herbal medicine for some aspect of primary health care [1]. Many of the species are used in traditional medicines for the treatment of a variety of diseases [2]. During the past several years, there has been an increase in the incidence of fungal infections due to the rise in immunocompromised population (e.g., organ transplant recipients and patients with cancer or human immunodeficiency virus infection/acquired immune deficiency syndrome). This fact coupled with the resistance to antibiotics and with toxicity during prolonged treatment with several antifungal drugs has been the reason for an extended search for newer drugs to treat opportunistic fungal infections. Infectious diseases, particularly skin and mucosal infections, are common in most of the tribal inhabitants due to lack of sanitation, potable water, and awareness of hygienic food habits. An important group of these skin pathogens is the fungi, among which dermatophytes and Candida spp. are prominent. Antimicrobial properties of certain Indian medicinal plants were reported based on folklore information and few studies were carried out on the inhibitory activity of these plants against certain pathogenic fungi [3]. Annona is the second largest genus of flowering plants in the family Annonaceae after Guatteria. Annona squamosa Linn., one of the important medicinal plants, commonly called “custard apple,” is a well-known plant of this family. It has been reported to possess a wide variety of pharmacological activities and is used in traditional applications [4]. A. squamosa is cultivated throughout India, America, Brazil, Southern Florida, and West Indies mainly for its edible fruits [5]. The plant A. squamosa Linn. is commonly called custard apple (English), sharifa (Hindi), sitappalam (Tamil), sita phalamu (Telugu), and sitaphala (Kannada) [4]. A. squamosa is a small tree that grows up to 3e8 m, with broad, irregularly spreading branches of light brown bark having thin leaves that occur singly, measuring 5e17 cm in length and 2e6 cm in width. Flowering (greenish yellow flowers on a hairy, slender 2-cm long stalk) occurs during the period from spring to early summer and flowers are pollinated by nitidulid beetles. The round or heart-shaped greenish yellow, ripened aggregate fruit is pendulous on a thickened stalk. The pulp of the fruit is white-tinged yellow, edible, and sweetly aromatic. Each carpel contains an oblong, shiny and smooth, dark brown to black, 1.3e1.6-cm long seed (Figure 1) [6]. Extensive biological research was carried out on this plant because of the presence of valuable annonaceous acetogenins in various parts of the plant, which are traditionally used for the treatment of many ailments [5]. So far, there are no systematic studies on the in vitro antifungal activity of the methanol, chloroform, and aqueous extracts as well as on the antioxidant effect of the chloroform extract of A. squamosa leaves. Therefore, this investigation was carried out to evaluate the antifungal and antioxidant potential of three different extracts of A. squamosa leaves.
Figure 1 e Annona squamosa Linn. plant: (A) flowers; (B) fruits; (C) seeds [7e10].
2.
Materials and methods
2.1.
Chemicals and solvents
All the chemicals used in this study were of analytical reagent grade and most of them were from S D Fine-Chem Ltd (India). Q3
2.2.
Collection of plant material
The leaves of A. squamosa were collected from fields of Dharwad, Karnataka, India, in February 2010. The leaves were identified and authenticated at the herbarium of the Botany Department of the Gulbarga University (voucher specimen HGUG No. 0019), Karnataka, India. After identification, the plant material was processed for extraction procedure.
2.3.
Preparation of the plant extract
The leaves of A. squamosa were thoroughly cleaned with water to remove dust particles and shade-dried at room temperature and reduced to coarse powder using a mechanical mixer. The powder was subjected to extraction by maceration using water, chloroform, and methanol to obtain their respective extracts. To 75 g of the powdered drug, 750 mL of solvent (water or chloroform or methanol) was added and stirred occasionally. The mixture was filtered on the 8th day, and the solvent was evaporated at 40 C to obtain a solid mass [11]. The percentage yields of water, chloroform, and methanol extracts were found to be 8.7% w/w, 10.1% w/w, and 12.6% w/w, respectively, which are stored in refrigerator (40 C) until further use.
2.4.
Preliminary phytochemical screening
All extracts were subjected to preliminary qualitative phytochemical screening to detect the presence of glycosides, alkaloids, amino acids, flavonoids, carbohydrates, saponins, phenols, steroids, and tannins by standard methods [12,13].
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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2.5.
Assessment of antifungal activity
Five pathogenic strains of fungi were used in the study, namely, Alternaria alternata, Candida albicans, Fusarium solani, Microsporum canis, and Aspergillus niger, which were collected from the Institute of Microbial Technology, Chandigarh, India. They were maintained in culture on Sabouraud glucose (4%) agar medium and used for antifungal assays as needed.
2.5.1.
Zone of inhibition of fungal growth
The antifungal activity of the leaf extracts was evaluated by the agar well diffusion method. The fungal inoculates were aseptically swabbed on sterile and solidified Sabouraud dextrose agar plates. Then, aseptically, 7-mm diameter wells were bored in the inoculated plates and the extracts [1 mg/mL and 2 mg/mL of 10% dimethyl sulfoxide (DMSO)], standard (ketoconazole 100 mg/mL), and blank (10% DMSO) were added separately into the respective labeled wells. The plates were incubated at 35 C for 72 hours in an upright position and the zone of inhibition formed around the well was recorded. The experiment was carried out in triplicates to get the average reading [14]. The percentage inhibition of the methanol, chloroform, and aqueous extracts of A. squamosa leaves was calculated using the following formula: % inhibition ¼ (diameter zone of sample/diameter zone of ketoconazole) 100 [15].
2.5.2. Minimum inhibitory concentration against different fungal strains The minimum inhibitory concentration (MIC) was determined using the broth dilution method. The fungal inoculum (105 dilution) was taken in test tubes with nutrient broth (1800 mL) supplemented with eight different concentrations (50 mg/mL, 100 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, 600 mg/mL, 800 mg/mL, and 1000 mg/mL) of all leaf extracts separately. The results of the extracts were compared with a standard, positive control (100 mg/mL ketoconazole), and negative control (respective solvent of each extract). All the test tubes were incubated at 35 C for 24e48 hours. The tubes were examined for visual turbidity. The MIC values were taken as the lowest concentration that inhibited the visual growth of the tested organisms [14,16].
2.6.
Assessment of antioxidant activity
2.6.1.
Radical scavenging activity
The half-maximal inhibitory concentration (IC50) values were determined as the concentration of the test mixture that gave 50% reduction in absorbance from that of the control.
2.6.1.1. DPPH free radical scavenging activity. The DPPH assay was performed as described by Koleva et al [17]. Approximately 10 mL of different concentrations (10e500 mg/mL) of test sample solutions were added to 190 mL DPPH (150 mM) in ethanol solution. The solutions were later vortex mixed and incubated for 20 minutes at 37 C. The solvent alone was considered “blank.” The decrease in absorbance of test mixtures (due to quenching of DPPH free radicals) was determined at 517 nm; ascorbic acid (5e250 mg/mL) was used as the standard [18]. 2.6.1.2. Hydrogen peroxide (H2O2) scavenging activity. To evaluate the hydrogen peroxide (H2O2) scavenging activity of the extracts, the H2O2 solution was prepared in phosphate buffer (pH 7.4) and then 0.6 mL of 40 mM H2O2 solution was mixed with 0.1 mL of different concentrations (10e500 mg/mL) of the extracts. Absorbance of hydrogen peroxide at 230 nm was determined after 10 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. Ascorbic acid (5e250 mg/mL) was taken as the reference compound [19]. 2.6.1.3. Nitric oxide radical scavenging activity. Nitric oxide (NO) generated from sodium nitroprusside was measured by the Griess reaction [20]. Sodium nitroprusside (5mM) in phosphate-buffered saline was mixed with 3 mL of different concentrations (10e500 mg/mL) of A. squamosa extracts and the mixture was incubated at 25 C for 150 minutes. The samples were then allowed to react with the Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2% H3PO4). The absorbance of the chromophore during the diazotization of nitrite with sulfanilamide and subsequent coupling with naphthyl ethylenediamine was measured at 546 nm. A similar procedure was repeated with respective solvent instead of the extract, which served as the control; Lascorbic acid (5e250 mg/mL) was used as the positive control [21]. All the tests were performed in triplicate. The percentage of scavenging activity was measured using the formula mentioned in the “Radical Scavenging Activity” section. 2.6.2.
The antioxidant activity was assessed through various in vitro assays. The free radical scavenging activities of the three extracts of A. squamosa leaves and L-ascorbic acid (vitamin C) were measured in terms of hydrogen donating or radical scavenging ability using the stable 1,1-diphenyl-2picrylhydrazyl (DPPH) radicals. Nitric acid generated from sodium nitroprusside was measured by the Griess reaction. The antioxidant activity was further confirmed by hydrogen peroxide (H2O2) scavenging and reducing power properties of A. squamosa leaves extracts. The percentage scavenging of free radical activity was calculated using the following formula: % inhibition ¼ [(Control e Test)/Control] 100
(1)
3
Reducing power property
The reducing power of extracts was determined according to the method suggested by Yen and Chen [22]. Different concentrations of each extract (25e500 mg/mL) were mixed with phosphate buffer (2.5 mL, 0.2M, pH 6.6) and potassium ferric cyanide [K3Fe(CN)6] (2.5 mL, 1%). The mixture was incubated at 50 C for 20 minutes. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture to stop the reaction, and the mixture was centrifuged at 3000 rpm for 10 minutes. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and ferric chloride (0.5 mL, 0.1%), and the absorbance was measured at 700 nm. A similar procedure was repeated with respective solvent instead of the extract, which served as the control. Ascorbic acid was used as the standard. All tests were performed in triplicate. Increased absorbance of the reaction mixture indicates the increased reducing power [23].
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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2.7.
Q4
Statistical analysis
The experimental results pertaining to the antifungal activity were expressed as the mean ± standard error of the mean (SEM), and for antioxidant activity, the average values from three parallel measurements were depicted in graphs. Linear regression analysis was used to calculate the IC50 values. Data were analyzed using one-way analysis of variance (ANOVA) followed by the Dunnett post hoc test using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).
3.
Results
3.1.
Phytochemical analysis
whereas against F. solani, the aqueous extract exhibited intermediate effect (Figure 2C) with percentage inhibition of 33.33% at 1 mg/mL and 64.58% at 2 mg/mL. Further, the methanolic and aqueous extracts showed maximum inhibitory activity against M. canis with percentage inhibition of 66.18% and 52.94% at 1 mg/mL and 72.06% and 67.65% at 2 mg/ mL of the methanol and aqueous extracts, respectively, when compared with the chloroform extract (Figure 2D) with percentage inhibition of 22.06% at 1 mg/mL and 41.18% at 2 mg/ mL. All the three extracts exhibited similar effect against A. niger with percentage inhibitions of 31.08%, 25.86%, and 22.41% at 1 mg/mL and 81.03%, 77.59%, and 84.84% at 2 mg/mL of methanol, chloroform, and aqueous extracts, respectively, as shown in the Figure 2E.
3.2.2. The phytochemical qualitative analysis revealed the presence of glycosides, flavonoids, phenols, tannins, saponins, alkaloids, carbohydrates, and steroids in different extracts as reported in Table 1.
3.2.
Antifungal assessment
3.2.1.
Agar well diffusion method
The results of this study revealed that the leaves of A. squamosa possess potential antifungal activity against all five pathogenic fungal strains studied. The antifungal activity was determined using the agar well diffusion method by measuring the diameter of zone of inhibition and by calculating the percentage inhibition. The antifungal activity of the tested extracts showed different selectivity for each microorganism studied. Our analysis indicated that both chloroform and methanol extracts showed almost similar effect against A. alternata, C. albicans, and F. solani (Figures 2Ae2C, respectively) with percentage inhibition of 79.10%, 27.69%, and 91.67% at 1 mg/mL and 83.58%, 64.62%, and 108.33% at 2 mg/mL of the chloroform extract, respectively, and 34.33%, 43.08%, and 60.42% at 1 mg/mL and 74.63%, 70.77%, and 97.92% at 2 mg/mL of the methanol extract, respectively. However, the aqueous extract of A. squamosa showed relatively less activity against A. alternata and C. albicans (Figures 2A and 2B, respectively), with percentage inhibition of 20.90% and 9.23% at 1 mg/mL and 43.28% and 33.85% at 2 mg/mL, respectively,
Table 1 e Preliminary phytochemical analysis of the methanol, chloroform, and aqueous extracts of Annona squamosa Linn. leaves. Extract
Methanol extract
Chloroform extract
Aqueous extract
þ þ þ þ þ þ þ e e
e þ þ þ e þ e e þ
e þ þ e þ þ þ þ e
Alkaloids Glycosides Flavonoids Tannins Carbohydrates Phenols Saponins Amino acids Steroids þ ¼ present; e ¼ absent.
Broth microdilution method
The MICs of methanol, chloroform, and aqueous extracts of A. squamosa leaves were estimated and presented in Table 2. The organic extracts exhibited significantly higher antifungal activity than the aqueous extract of A. squamosa leaves. The chloroform extract showed maximum potency among the three extracts tested against A. alternata and F. solani. The methanol extract exhibited the highest activity against M. canis and A. niger; however, against C. albicans both methanol and chloroform extracts showed the same potency. The aqueous extract showed relatively less activity when compared with the methanol and chloroform extracts against all tested fungal strains.
3.3.
Antioxidant assessment
3.3.1.
Radical scavenging activity
The results pertaining to the DPPH free radical scavenging activity of the three different extracts of A. squamosa leaves, along with the standard reference, vitamin C, are shown in Figure 3A. The potent free radical scavenging activity was exhibited by the chloroform extract (IC50 308.3 mg/mL), whereas the methanol extract (IC50 342.5 mg/mL) showed comparable scavenging activity and the aqueous extract (IC50 439.6 mg/mL) exhibited relatively less free radical scavenging activity. Ascorbic acid showed the highest DPPH radical scavenging activity (IC50 35.26 mg/mL). Figure 3B shows hydrogen peroxide scavenging activity of the methanol, chloroform, and aqueous extracts of A. squamosa leaves. All extracts caused a strong dose-dependent inhibition of hydrogen peroxide. The aqueous and chloroform extracts showed good H2O2 scavenging ability compared with the methanol extract. The IC50 values of the chloroform, aqueous, and methanol extracts were 242.7 mg/mL, 342.2 mg/ mL, and 453.7 mg/mL, respectively; however, ascorbic acid showed maximum activity with IC50 of 32.73 mg/mL. Results related to the NO radical scavenging activity of all the extracts and positive control showed significant NO radical scavenging activity in a concentration-dependent manner (Figure 3C). In this assay, the methanol extract caused a potential inhibition when compared with the chloroform and aqueous extracts of A. squamosa leaves with IC50 values of 185.2 mg/mL, 345.8 mg/mL, and 366.3 mg/mL, respectively. The IC50 value of ascorbic acid was 58.7 mg/mL.
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Figure 2 e (A) Antifungal activity of Annona squamosa Linn. against Alternaria alternata. (B) Antifungal activity of A. squamosa against Candida albicans. (C) Antifungal activity of A. squamosa against Fusarium solani. (D) Antifungal activity of A. squamosa against Microsporum canis. (E) Antifungal activity of A. squamosa against Aspergillus niger. The values are expressed as mean ± standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.01 versus vehicle control (blank), calculated using oneway analysis of variance followed by Dunnett post hoc analysis (n ¼ 3). AEAS ¼ aqueous extract; CEAS ¼ chloroform extract; KTZ ¼ ketoconazole; MEAS ¼ methanol extract.
3.3.2.
Reducing power assay
The reducing power property of the methanol, chloroform, and aqueous extracts is illustrated in Figure 4. In the reductive ability measurement, Fe3þ to Fe2þ transformation in the presence of extract sample was investigated. In this study, the reductive capabilities of the chloroform, methanol, and aqueous extracts of A. squamosa leaves were increased with increase in their concentration.
4.
Discussion
Antimicrobial activities of plant leaves, flowers, stems, roots, or fruits from various herbs and spices have been reported by many workers [24]. Successful prediction of botanical compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Results of the
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Table 2 e Minimum inhibitory concentrations of different extracts of Annona squamosa Linn. against various fungal strains. Fungus Alternaria alternata Candia albicans Fusarium solani Microsporum canis Aspergillus niger
Methanol Chloroform Aqueous extract (mg/mL) extract (mg/mL) extract (mg/mL) 800
200
1000
600
600
800
600
300
800
400
800
600
400
600
1000
antifungal assay carried out with aqueous and organic extracts of A. squamosa leaves showed their inhibitory effect at both concentrations tested (1 mg/mL and 2 mg/mL) against the five pathogenic fungi in a dose-dependent manner. These pathogens included A. niger, M. canis, F. solani, A. alternata, and C. albicans, which are known to cause aspergillosis, dermatophytosis, Fusarium keratitis, respiratory tract infections, and candidiasis, respectively. The antifungal activity was higher
for the methanol and chloroform extracts against A. alternata, C. albicans, and F. solani, whereas the methanol and aqueous extracts showed better effect than the chloroform extract against M. canis, which causes tinea corporis and tinea capitis in humans. However, all the tested extracts showed similar inhibitory activity against the fungus A. niger, which is a filamentous ascomycete that is ubiquitous in the environment and has been implicated in opportunistic infections of humans. It causes various diseases in plants and animals. In plants, it causes black mold and rot diseases and in human beings it causes aspergillosis, which leads to pulmonary allergy, bronchopulmonary aspergillosis, and pulmonary aspergilloma [16]. The presented result may further indicate that the antimicrobial properties/chemical constituents, which are either polar or nonpolar, might be effectively extracted through the organic solvent medium. Some of the earlier studies also reported the effective inhibitory activity of alcoholic solvents against the growth of pathogenic fungi. The aqueous (water) extract showed a much lesser inhibitory effect, which might be attributed to the extracting capacity of solvent and the concentration of the active ingredients in the extracts; in addition, most of the active ingredients dissolve better in alcoholic solvents than in water. Several researchers have reported that aqueous (water) extracts do not exhibit
Figure 3 e (A) 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity of Annona squamosa Linn. leaves. (B) H2O2 scavenging activity of A. squamosa leaves. (C) Nitric oxide radical scavenging activity of A. squamosa leaves. AEAS ¼ aqueous extract; CEAS ¼ chloroform extract; MEAS ¼ methanol extract. Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Figure 4 e Reducing power property of Annona squamosa Linn. leaves extracts. AEAS ¼ aqueous extract; CEAS ¼ chloroform extract; MEAS ¼ methanol extract.
much activity against fungi [25]. Further, the evaluation of minimum inhibitory concentration of A. squamosa leaves extracts by the broth dilution method revealed the differential potency of each extract against the tested fungal strains. The most susceptible species for the chloroform extract of A. squamosa leaves were A. alternata and F. solani, whereas C. albicans, M. canis, and A. niger were found to be more susceptible to the methanol extract of A. squamosa The varying degree of sensitivity of tested fungi may be due to the intrinsic tolerance of microorganisms and the nature and combinations of phytocompounds present in the crude extracts. The bioactivity of plant extracts was attributed to the presence of phytochemical constituents; for example, plants rich in saponins have immune-boosting, anti-inflammatory [26], antiviral, and antibacterial properties [27]. Similarly, tannins have been reported to have antibacterial potential due to their basic character that allows them to react with proteins to form stable water-soluble compounds, thereby killing bacteria by directly damaging their cell membrane. Flavonoids are a major group of phenolic compounds reported for their antiviral, antimicrobial, and spasmolytic properties [26]. Alkaloids isolated from plants are commonly found to have antimicrobial properties [28]. With regard to natural products, it is generally accepted that phytochemicals are less potent antiinfectives than agents of microbial origin (i.e., antibiotics). However, new classes of antimicrobial drugs are urgently required and the flavonoids represent a novel set of leads. Future optimization of these compounds through structural alteration may allow the development of a pharmacologically acceptable antimicrobial agent or group of agents. The researchers reported that screening of these analogs might lead to the identification of compounds that are sufficiently potent to be useful as antifungal, antiviral or antibacterial chemotherapeutics [29]. The antifungal activity of the leaves extracts of A. squamosa as recorded in this study may be attributed to the presence of flavonoids; however, the higher degree of antifungal potency of the chloroform and methanol extracts recorded in this study might be due to the presence of tannins. Whereas the isolation and characterization of medicinally
7
important bioactive phytoconstituents from the leaves of A. squamosa are required for the development of potential antifungal agents, our report provides a strong evidence of the ability of the methanolic and chloroform extracts of A. squamosa leaves against the fungal species tested. The DPPH free radical scavenging model can be used to evaluate the antioxidant activity in a relatively short time. DPPH is a stable free radical and accepts either an electron or OH* to become a stable diamagnetic molecule [30]. The decreased absorbance results in a color change from purple to yellow, as radicals were scavenged by antioxidants through the donation of hydrogen to form the stable DPPH molecule [31]. Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of the essential thiol groups. Hydrogen peroxide crosses cell membrane and reacts with ferric and copper ions, which show toxic effects [32]. Similarly, the reactive oxygen species NO is also implicated in inflammation, cancer, and other pathological conditions [33]. NO is a very unstable species under aerobic conditions [34]. The plant products have the property to counteract the effect of NO formation and, in turn, may be of considerable interest in relation to preventing the ill effects of excessive NO generation in the human body. Several studies have evaluated the relationship between the antioxidant activity of plant products and their phenolic content. Substances that are able to perform these reactions can be considered as antioxidants and radical scavengers [18]. Reducing power, which was used to measure the reductive ability of antioxidants, was evaluated by the transformation of Fe3þ to Fe2þ in the presence of the leaves extracts [35]. Antioxidants reduce the Fe3þ/ferricyanide complex to the ferrous form by donating an electron. The color of the test solution then changes from yellow to different shades of green and blue [36]. The ability of these extracts to reduce Fe3þ may be attributed to the hydrogen donating effect of phenolic compounds [37]. It has been reported that phenolic compounds were the main antioxidant components, and their total contents were directly proportional to their antioxidant activity [38]. Therefore, in this study, the presence of the flavonoids and phenols in all the tested extracts of A. squamosa leaves might have contributed to the antioxidant activity.
5.
Conclusion
From the aforementioned results, it can be concluded that the chloroform, methanol, and aqueous extracts of the leaves of A. squamosa have in vitro antifungal and antioxidant activities. Further studies are required to isolate the active components from the extracts and to elucidate the exact mechanism of action of the antifungal and antioxidant activities.
Conflicts of interest The authors declare that there are no conflicts of interest.
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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references
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Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Original Article
Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves Q5
Narasimharaju Kalidindi a,*, Nagarasanakote Venkataswamy Thimmaiah b, Nagepally Venkataramareddy Jagadeesh c, Ramaiah Nandeep a, Sathyanarayana Swetha a, Bharat Kalidindi d a
Department of Pharmacology, Visveswarapura Institute of Pharmaceutical Sciences, Bangalore, Karnataka, India Department of Pharmacology, KLE College of Pharmacy, Belgaum, Karnataka, India c Department of Pharmacology, SET's College of Pharmacy, Dharwad, Karnataka, India d SDM College of Ayurveda Kuthpady, Udupi, Karnataka, India b
article info
abstract
Article history:
An increasing demand for natural additives has shifted the attention from synthetic to
Received 1 November 2014
natural antioxidants and antifungal agents. This study was carried out to evaluate the
Received in revised form
antifungal and antioxidant activities of methanol, chloroform, and aqueous extracts of
15 April 2015
Annona squamosa Linn. leaves. The antifungal activities of all extracts of A. squamosa leaves
Accepted 26 April 2015
against five different strains of fungi (Alternaria alternata, Candida albicans, Fusarium solani,
Available online xxx
Microsporum canis, and Aspergillus niger) were evaluated by the agar well diffusion method and the minimum inhibitory concentration of each extract was assessed by antifungal suscep-
Q1
Keywords:
tibility using the broth microdilution method. The antioxidant potential of each extract was
acetogenins
determined by free radicals (1,1-diphenyl-2-picrylhydrazyl, nitric oxide, and hydrogen
Annona
peroxide) scavenging activity and reducing power property of A. squamosa leaves. Both
antifungal
organic and aqueous extracts were found to express dose-dependent inhibition against all
antioxidant
tested fungi strains in both agar well diffusion and broth dilution methods. The free radical
extract
scavenging activity and reducing power property of all extracts were found to be concentration dependent, with the methanol extract exhibiting higher antioxidant activity than the chloroform extract, which was more effective than the aqueous extract of A. squamosa leaves. Results of phytochemical analysis of extracts showed the presence of glycosides, saponins, tannins, flavonoids, phenols, etc. The results obtained from in vitro studies of antifungal and antioxidant activities clearly suggest that the methanol, chloroform, and aqueous extracts of A. squamosa leaves possess antifungal and antioxidant activity. Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.
* Corresponding author. Department of Pharmacology, Visveswarapura Institute of Pharmaceutical Sciences, 22nd Main, 24th Cross, Banashankari II Stage, Bangalore 560 070, Karnataka, India. E-mail address: [email protected] (N. Kalidindi). http://dx.doi.org/10.1016/j.jfda.2015.04.012 1021-9498/Copyright © 2015, Food and Drug Administration, Taiwan. Published by Elsevier Taiwan LLC. All rights reserved.
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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1. Q2
j o u r n a l o f f o o d a n d d r u g a n a l y s i s x x x ( 2 0 1 5 ) 1 e8
Introduction
In modern times, the use of herbal products has significantly increased in the developed countries as well as in several other countries. According to a World Health Organization estimate, 80% of the world's population presently uses herbal medicine for some aspect of primary health care [1]. Many of the species are used in traditional medicines for the treatment of a variety of diseases [2]. During the past several years, there has been an increase in the incidence of fungal infections due to the rise in immunocompromised population (e.g., organ transplant recipients and patients with cancer or human immunodeficiency virus infection/acquired immune deficiency syndrome). This fact coupled with the resistance to antibiotics and with toxicity during prolonged treatment with several antifungal drugs has been the reason for an extended search for newer drugs to treat opportunistic fungal infections. Infectious diseases, particularly skin and mucosal infections, are common in most of the tribal inhabitants due to lack of sanitation, potable water, and awareness of hygienic food habits. An important group of these skin pathogens is the fungi, among which dermatophytes and Candida spp. are prominent. Antimicrobial properties of certain Indian medicinal plants were reported based on folklore information and few studies were carried out on the inhibitory activity of these plants against certain pathogenic fungi [3]. Annona is the second largest genus of flowering plants in the family Annonaceae after Guatteria. Annona squamosa Linn., one of the important medicinal plants, commonly called “custard apple,” is a well-known plant of this family. It has been reported to possess a wide variety of pharmacological activities and is used in traditional applications [4]. A. squamosa is cultivated throughout India, America, Brazil, Southern Florida, and West Indies mainly for its edible fruits [5]. The plant A. squamosa Linn. is commonly called custard apple (English), sharifa (Hindi), sitappalam (Tamil), sita phalamu (Telugu), and sitaphala (Kannada) [4]. A. squamosa is a small tree that grows up to 3e8 m, with broad, irregularly spreading branches of light brown bark having thin leaves that occur singly, measuring 5e17 cm in length and 2e6 cm in width. Flowering (greenish yellow flowers on a hairy, slender 2-cm long stalk) occurs during the period from spring to early summer and flowers are pollinated by nitidulid beetles. The round or heart-shaped greenish yellow, ripened aggregate fruit is pendulous on a thickened stalk. The pulp of the fruit is white-tinged yellow, edible, and sweetly aromatic. Each carpel contains an oblong, shiny and smooth, dark brown to black, 1.3e1.6-cm long seed (Figure 1) [6]. Extensive biological research was carried out on this plant because of the presence of valuable annonaceous acetogenins in various parts of the plant, which are traditionally used for the treatment of many ailments [5]. So far, there are no systematic studies on the in vitro antifungal activity of the methanol, chloroform, and aqueous extracts as well as on the antioxidant effect of the chloroform extract of A. squamosa leaves. Therefore, this investigation was carried out to evaluate the antifungal and antioxidant potential of three different extracts of A. squamosa leaves.
Figure 1 e Annona squamosa Linn. plant: (A) flowers; (B) fruits; (C) seeds [7e10].
2.
Materials and methods
2.1.
Chemicals and solvents
All the chemicals used in this study were of analytical reagent grade and most of them were from S D Fine-Chem Ltd (India). Q3
2.2.
Collection of plant material
The leaves of A. squamosa were collected from fields of Dharwad, Karnataka, India, in February 2010. The leaves were identified and authenticated at the herbarium of the Botany Department of the Gulbarga University (voucher specimen HGUG No. 0019), Karnataka, India. After identification, the plant material was processed for extraction procedure.
2.3.
Preparation of the plant extract
The leaves of A. squamosa were thoroughly cleaned with water to remove dust particles and shade-dried at room temperature and reduced to coarse powder using a mechanical mixer. The powder was subjected to extraction by maceration using water, chloroform, and methanol to obtain their respective extracts. To 75 g of the powdered drug, 750 mL of solvent (water or chloroform or methanol) was added and stirred occasionally. The mixture was filtered on the 8th day, and the solvent was evaporated at 40 C to obtain a solid mass [11]. The percentage yields of water, chloroform, and methanol extracts were found to be 8.7% w/w, 10.1% w/w, and 12.6% w/w, respectively, which are stored in refrigerator (40 C) until further use.
2.4.
Preliminary phytochemical screening
All extracts were subjected to preliminary qualitative phytochemical screening to detect the presence of glycosides, alkaloids, amino acids, flavonoids, carbohydrates, saponins, phenols, steroids, and tannins by standard methods [12,13].
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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2.5.
Assessment of antifungal activity
Five pathogenic strains of fungi were used in the study, namely, Alternaria alternata, Candida albicans, Fusarium solani, Microsporum canis, and Aspergillus niger, which were collected from the Institute of Microbial Technology, Chandigarh, India. They were maintained in culture on Sabouraud glucose (4%) agar medium and used for antifungal assays as needed.
2.5.1.
Zone of inhibition of fungal growth
The antifungal activity of the leaf extracts was evaluated by the agar well diffusion method. The fungal inoculates were aseptically swabbed on sterile and solidified Sabouraud dextrose agar plates. Then, aseptically, 7-mm diameter wells were bored in the inoculated plates and the extracts [1 mg/mL and 2 mg/mL of 10% dimethyl sulfoxide (DMSO)], standard (ketoconazole 100 mg/mL), and blank (10% DMSO) were added separately into the respective labeled wells. The plates were incubated at 35 C for 72 hours in an upright position and the zone of inhibition formed around the well was recorded. The experiment was carried out in triplicates to get the average reading [14]. The percentage inhibition of the methanol, chloroform, and aqueous extracts of A. squamosa leaves was calculated using the following formula: % inhibition ¼ (diameter zone of sample/diameter zone of ketoconazole) 100 [15].
2.5.2. Minimum inhibitory concentration against different fungal strains The minimum inhibitory concentration (MIC) was determined using the broth dilution method. The fungal inoculum (105 dilution) was taken in test tubes with nutrient broth (1800 mL) supplemented with eight different concentrations (50 mg/mL, 100 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, 600 mg/mL, 800 mg/mL, and 1000 mg/mL) of all leaf extracts separately. The results of the extracts were compared with a standard, positive control (100 mg/mL ketoconazole), and negative control (respective solvent of each extract). All the test tubes were incubated at 35 C for 24e48 hours. The tubes were examined for visual turbidity. The MIC values were taken as the lowest concentration that inhibited the visual growth of the tested organisms [14,16].
2.6.
Assessment of antioxidant activity
2.6.1.
Radical scavenging activity
The half-maximal inhibitory concentration (IC50) values were determined as the concentration of the test mixture that gave 50% reduction in absorbance from that of the control.
2.6.1.1. DPPH free radical scavenging activity. The DPPH assay was performed as described by Koleva et al [17]. Approximately 10 mL of different concentrations (10e500 mg/mL) of test sample solutions were added to 190 mL DPPH (150 mM) in ethanol solution. The solutions were later vortex mixed and incubated for 20 minutes at 37 C. The solvent alone was considered “blank.” The decrease in absorbance of test mixtures (due to quenching of DPPH free radicals) was determined at 517 nm; ascorbic acid (5e250 mg/mL) was used as the standard [18]. 2.6.1.2. Hydrogen peroxide (H2O2) scavenging activity. To evaluate the hydrogen peroxide (H2O2) scavenging activity of the extracts, the H2O2 solution was prepared in phosphate buffer (pH 7.4) and then 0.6 mL of 40 mM H2O2 solution was mixed with 0.1 mL of different concentrations (10e500 mg/mL) of the extracts. Absorbance of hydrogen peroxide at 230 nm was determined after 10 minutes against a blank solution containing phosphate buffer without hydrogen peroxide. Ascorbic acid (5e250 mg/mL) was taken as the reference compound [19]. 2.6.1.3. Nitric oxide radical scavenging activity. Nitric oxide (NO) generated from sodium nitroprusside was measured by the Griess reaction [20]. Sodium nitroprusside (5mM) in phosphate-buffered saline was mixed with 3 mL of different concentrations (10e500 mg/mL) of A. squamosa extracts and the mixture was incubated at 25 C for 150 minutes. The samples were then allowed to react with the Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2% H3PO4). The absorbance of the chromophore during the diazotization of nitrite with sulfanilamide and subsequent coupling with naphthyl ethylenediamine was measured at 546 nm. A similar procedure was repeated with respective solvent instead of the extract, which served as the control; Lascorbic acid (5e250 mg/mL) was used as the positive control [21]. All the tests were performed in triplicate. The percentage of scavenging activity was measured using the formula mentioned in the “Radical Scavenging Activity” section. 2.6.2.
The antioxidant activity was assessed through various in vitro assays. The free radical scavenging activities of the three extracts of A. squamosa leaves and L-ascorbic acid (vitamin C) were measured in terms of hydrogen donating or radical scavenging ability using the stable 1,1-diphenyl-2picrylhydrazyl (DPPH) radicals. Nitric acid generated from sodium nitroprusside was measured by the Griess reaction. The antioxidant activity was further confirmed by hydrogen peroxide (H2O2) scavenging and reducing power properties of A. squamosa leaves extracts. The percentage scavenging of free radical activity was calculated using the following formula: % inhibition ¼ [(Control e Test)/Control] 100
(1)
3
Reducing power property
The reducing power of extracts was determined according to the method suggested by Yen and Chen [22]. Different concentrations of each extract (25e500 mg/mL) were mixed with phosphate buffer (2.5 mL, 0.2M, pH 6.6) and potassium ferric cyanide [K3Fe(CN)6] (2.5 mL, 1%). The mixture was incubated at 50 C for 20 minutes. A portion (2.5 mL) of trichloroacetic acid (10%) was added to the mixture to stop the reaction, and the mixture was centrifuged at 3000 rpm for 10 minutes. The upper layer of solution (2.5 mL) was mixed with distilled water (2.5 mL) and ferric chloride (0.5 mL, 0.1%), and the absorbance was measured at 700 nm. A similar procedure was repeated with respective solvent instead of the extract, which served as the control. Ascorbic acid was used as the standard. All tests were performed in triplicate. Increased absorbance of the reaction mixture indicates the increased reducing power [23].
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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j o u r n a l o f f o o d a n d d r u g a n a l y s i s x x x ( 2 0 1 5 ) 1 e8
2.7.
Q4
Statistical analysis
The experimental results pertaining to the antifungal activity were expressed as the mean ± standard error of the mean (SEM), and for antioxidant activity, the average values from three parallel measurements were depicted in graphs. Linear regression analysis was used to calculate the IC50 values. Data were analyzed using one-way analysis of variance (ANOVA) followed by the Dunnett post hoc test using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).
3.
Results
3.1.
Phytochemical analysis
whereas against F. solani, the aqueous extract exhibited intermediate effect (Figure 2C) with percentage inhibition of 33.33% at 1 mg/mL and 64.58% at 2 mg/mL. Further, the methanolic and aqueous extracts showed maximum inhibitory activity against M. canis with percentage inhibition of 66.18% and 52.94% at 1 mg/mL and 72.06% and 67.65% at 2 mg/ mL of the methanol and aqueous extracts, respectively, when compared with the chloroform extract (Figure 2D) with percentage inhibition of 22.06% at 1 mg/mL and 41.18% at 2 mg/ mL. All the three extracts exhibited similar effect against A. niger with percentage inhibitions of 31.08%, 25.86%, and 22.41% at 1 mg/mL and 81.03%, 77.59%, and 84.84% at 2 mg/mL of methanol, chloroform, and aqueous extracts, respectively, as shown in the Figure 2E.
3.2.2. The phytochemical qualitative analysis revealed the presence of glycosides, flavonoids, phenols, tannins, saponins, alkaloids, carbohydrates, and steroids in different extracts as reported in Table 1.
3.2.
Antifungal assessment
3.2.1.
Agar well diffusion method
The results of this study revealed that the leaves of A. squamosa possess potential antifungal activity against all five pathogenic fungal strains studied. The antifungal activity was determined using the agar well diffusion method by measuring the diameter of zone of inhibition and by calculating the percentage inhibition. The antifungal activity of the tested extracts showed different selectivity for each microorganism studied. Our analysis indicated that both chloroform and methanol extracts showed almost similar effect against A. alternata, C. albicans, and F. solani (Figures 2Ae2C, respectively) with percentage inhibition of 79.10%, 27.69%, and 91.67% at 1 mg/mL and 83.58%, 64.62%, and 108.33% at 2 mg/mL of the chloroform extract, respectively, and 34.33%, 43.08%, and 60.42% at 1 mg/mL and 74.63%, 70.77%, and 97.92% at 2 mg/mL of the methanol extract, respectively. However, the aqueous extract of A. squamosa showed relatively less activity against A. alternata and C. albicans (Figures 2A and 2B, respectively), with percentage inhibition of 20.90% and 9.23% at 1 mg/mL and 43.28% and 33.85% at 2 mg/mL, respectively,
Table 1 e Preliminary phytochemical analysis of the methanol, chloroform, and aqueous extracts of Annona squamosa Linn. leaves. Extract
Methanol extract
Chloroform extract
Aqueous extract
þ þ þ þ þ þ þ e e
e þ þ þ e þ e e þ
e þ þ e þ þ þ þ e
Alkaloids Glycosides Flavonoids Tannins Carbohydrates Phenols Saponins Amino acids Steroids þ ¼ present; e ¼ absent.
Broth microdilution method
The MICs of methanol, chloroform, and aqueous extracts of A. squamosa leaves were estimated and presented in Table 2. The organic extracts exhibited significantly higher antifungal activity than the aqueous extract of A. squamosa leaves. The chloroform extract showed maximum potency among the three extracts tested against A. alternata and F. solani. The methanol extract exhibited the highest activity against M. canis and A. niger; however, against C. albicans both methanol and chloroform extracts showed the same potency. The aqueous extract showed relatively less activity when compared with the methanol and chloroform extracts against all tested fungal strains.
3.3.
Antioxidant assessment
3.3.1.
Radical scavenging activity
The results pertaining to the DPPH free radical scavenging activity of the three different extracts of A. squamosa leaves, along with the standard reference, vitamin C, are shown in Figure 3A. The potent free radical scavenging activity was exhibited by the chloroform extract (IC50 308.3 mg/mL), whereas the methanol extract (IC50 342.5 mg/mL) showed comparable scavenging activity and the aqueous extract (IC50 439.6 mg/mL) exhibited relatively less free radical scavenging activity. Ascorbic acid showed the highest DPPH radical scavenging activity (IC50 35.26 mg/mL). Figure 3B shows hydrogen peroxide scavenging activity of the methanol, chloroform, and aqueous extracts of A. squamosa leaves. All extracts caused a strong dose-dependent inhibition of hydrogen peroxide. The aqueous and chloroform extracts showed good H2O2 scavenging ability compared with the methanol extract. The IC50 values of the chloroform, aqueous, and methanol extracts were 242.7 mg/mL, 342.2 mg/ mL, and 453.7 mg/mL, respectively; however, ascorbic acid showed maximum activity with IC50 of 32.73 mg/mL. Results related to the NO radical scavenging activity of all the extracts and positive control showed significant NO radical scavenging activity in a concentration-dependent manner (Figure 3C). In this assay, the methanol extract caused a potential inhibition when compared with the chloroform and aqueous extracts of A. squamosa leaves with IC50 values of 185.2 mg/mL, 345.8 mg/mL, and 366.3 mg/mL, respectively. The IC50 value of ascorbic acid was 58.7 mg/mL.
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Figure 2 e (A) Antifungal activity of Annona squamosa Linn. against Alternaria alternata. (B) Antifungal activity of A. squamosa against Candida albicans. (C) Antifungal activity of A. squamosa against Fusarium solani. (D) Antifungal activity of A. squamosa against Microsporum canis. (E) Antifungal activity of A. squamosa against Aspergillus niger. The values are expressed as mean ± standard error of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.01 versus vehicle control (blank), calculated using oneway analysis of variance followed by Dunnett post hoc analysis (n ¼ 3). AEAS ¼ aqueous extract; CEAS ¼ chloroform extract; KTZ ¼ ketoconazole; MEAS ¼ methanol extract.
3.3.2.
Reducing power assay
The reducing power property of the methanol, chloroform, and aqueous extracts is illustrated in Figure 4. In the reductive ability measurement, Fe3þ to Fe2þ transformation in the presence of extract sample was investigated. In this study, the reductive capabilities of the chloroform, methanol, and aqueous extracts of A. squamosa leaves were increased with increase in their concentration.
4.
Discussion
Antimicrobial activities of plant leaves, flowers, stems, roots, or fruits from various herbs and spices have been reported by many workers [24]. Successful prediction of botanical compounds from plant material is largely dependent on the type of solvent used in the extraction procedure. Results of the
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Table 2 e Minimum inhibitory concentrations of different extracts of Annona squamosa Linn. against various fungal strains. Fungus Alternaria alternata Candia albicans Fusarium solani Microsporum canis Aspergillus niger
Methanol Chloroform Aqueous extract (mg/mL) extract (mg/mL) extract (mg/mL) 800
200
1000
600
600
800
600
300
800
400
800
600
400
600
1000
antifungal assay carried out with aqueous and organic extracts of A. squamosa leaves showed their inhibitory effect at both concentrations tested (1 mg/mL and 2 mg/mL) against the five pathogenic fungi in a dose-dependent manner. These pathogens included A. niger, M. canis, F. solani, A. alternata, and C. albicans, which are known to cause aspergillosis, dermatophytosis, Fusarium keratitis, respiratory tract infections, and candidiasis, respectively. The antifungal activity was higher
for the methanol and chloroform extracts against A. alternata, C. albicans, and F. solani, whereas the methanol and aqueous extracts showed better effect than the chloroform extract against M. canis, which causes tinea corporis and tinea capitis in humans. However, all the tested extracts showed similar inhibitory activity against the fungus A. niger, which is a filamentous ascomycete that is ubiquitous in the environment and has been implicated in opportunistic infections of humans. It causes various diseases in plants and animals. In plants, it causes black mold and rot diseases and in human beings it causes aspergillosis, which leads to pulmonary allergy, bronchopulmonary aspergillosis, and pulmonary aspergilloma [16]. The presented result may further indicate that the antimicrobial properties/chemical constituents, which are either polar or nonpolar, might be effectively extracted through the organic solvent medium. Some of the earlier studies also reported the effective inhibitory activity of alcoholic solvents against the growth of pathogenic fungi. The aqueous (water) extract showed a much lesser inhibitory effect, which might be attributed to the extracting capacity of solvent and the concentration of the active ingredients in the extracts; in addition, most of the active ingredients dissolve better in alcoholic solvents than in water. Several researchers have reported that aqueous (water) extracts do not exhibit
Figure 3 e (A) 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity of Annona squamosa Linn. leaves. (B) H2O2 scavenging activity of A. squamosa leaves. (C) Nitric oxide radical scavenging activity of A. squamosa leaves. AEAS ¼ aqueous extract; CEAS ¼ chloroform extract; MEAS ¼ methanol extract. Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Figure 4 e Reducing power property of Annona squamosa Linn. leaves extracts. AEAS ¼ aqueous extract; CEAS ¼ chloroform extract; MEAS ¼ methanol extract.
much activity against fungi [25]. Further, the evaluation of minimum inhibitory concentration of A. squamosa leaves extracts by the broth dilution method revealed the differential potency of each extract against the tested fungal strains. The most susceptible species for the chloroform extract of A. squamosa leaves were A. alternata and F. solani, whereas C. albicans, M. canis, and A. niger were found to be more susceptible to the methanol extract of A. squamosa The varying degree of sensitivity of tested fungi may be due to the intrinsic tolerance of microorganisms and the nature and combinations of phytocompounds present in the crude extracts. The bioactivity of plant extracts was attributed to the presence of phytochemical constituents; for example, plants rich in saponins have immune-boosting, anti-inflammatory [26], antiviral, and antibacterial properties [27]. Similarly, tannins have been reported to have antibacterial potential due to their basic character that allows them to react with proteins to form stable water-soluble compounds, thereby killing bacteria by directly damaging their cell membrane. Flavonoids are a major group of phenolic compounds reported for their antiviral, antimicrobial, and spasmolytic properties [26]. Alkaloids isolated from plants are commonly found to have antimicrobial properties [28]. With regard to natural products, it is generally accepted that phytochemicals are less potent antiinfectives than agents of microbial origin (i.e., antibiotics). However, new classes of antimicrobial drugs are urgently required and the flavonoids represent a novel set of leads. Future optimization of these compounds through structural alteration may allow the development of a pharmacologically acceptable antimicrobial agent or group of agents. The researchers reported that screening of these analogs might lead to the identification of compounds that are sufficiently potent to be useful as antifungal, antiviral or antibacterial chemotherapeutics [29]. The antifungal activity of the leaves extracts of A. squamosa as recorded in this study may be attributed to the presence of flavonoids; however, the higher degree of antifungal potency of the chloroform and methanol extracts recorded in this study might be due to the presence of tannins. Whereas the isolation and characterization of medicinally
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important bioactive phytoconstituents from the leaves of A. squamosa are required for the development of potential antifungal agents, our report provides a strong evidence of the ability of the methanolic and chloroform extracts of A. squamosa leaves against the fungal species tested. The DPPH free radical scavenging model can be used to evaluate the antioxidant activity in a relatively short time. DPPH is a stable free radical and accepts either an electron or OH* to become a stable diamagnetic molecule [30]. The decreased absorbance results in a color change from purple to yellow, as radicals were scavenged by antioxidants through the donation of hydrogen to form the stable DPPH molecule [31]. Hydrogen peroxide is a weak oxidizing agent and can inactivate a few enzymes directly, usually by oxidation of the essential thiol groups. Hydrogen peroxide crosses cell membrane and reacts with ferric and copper ions, which show toxic effects [32]. Similarly, the reactive oxygen species NO is also implicated in inflammation, cancer, and other pathological conditions [33]. NO is a very unstable species under aerobic conditions [34]. The plant products have the property to counteract the effect of NO formation and, in turn, may be of considerable interest in relation to preventing the ill effects of excessive NO generation in the human body. Several studies have evaluated the relationship between the antioxidant activity of plant products and their phenolic content. Substances that are able to perform these reactions can be considered as antioxidants and radical scavengers [18]. Reducing power, which was used to measure the reductive ability of antioxidants, was evaluated by the transformation of Fe3þ to Fe2þ in the presence of the leaves extracts [35]. Antioxidants reduce the Fe3þ/ferricyanide complex to the ferrous form by donating an electron. The color of the test solution then changes from yellow to different shades of green and blue [36]. The ability of these extracts to reduce Fe3þ may be attributed to the hydrogen donating effect of phenolic compounds [37]. It has been reported that phenolic compounds were the main antioxidant components, and their total contents were directly proportional to their antioxidant activity [38]. Therefore, in this study, the presence of the flavonoids and phenols in all the tested extracts of A. squamosa leaves might have contributed to the antioxidant activity.
5.
Conclusion
From the aforementioned results, it can be concluded that the chloroform, methanol, and aqueous extracts of the leaves of A. squamosa have in vitro antifungal and antioxidant activities. Further studies are required to isolate the active components from the extracts and to elucidate the exact mechanism of action of the antifungal and antioxidant activities.
Conflicts of interest The authors declare that there are no conflicts of interest.
Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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Please cite this article in press as: Kalidindi N, et al., Antifungal and antioxidant activities of organic and aqueous extracts of Annona squamosa Linn. leaves, Journal of Food and Drug Analysis (2015), http://dx.doi.org/10.1016/j.jfda.2015.04.012
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